We will continue purification and characterization of lymphotoxins (LTS) derived from PMA-stimulated human B-lymphoblastoid cell lines. Purification of alpha-LT from the RPMI 1788 line will be acomplished with one final anticipated step. The electrophoretically homogeneous product will be analyzed by SDS-PAGE. Two models for the structure of the 70-90kd alpha-LT molecule will be examined: a) The alpha-LT is a multimer of a 20kd subunit, as suggested by evidence obtained with currently available cDNA or, b) the alpha-LT is a heterodimer of the 20kd subunit and a 70kd subunit, as suggested by several studies analyzing purified LTs derived from lectin-stimulated human lymphocytes or from a B-lymphoblastoid cell line. If our evidence using the RPMI 1788 alpha-LT further supports b) and the multi-gene nature of the LT system we will i.) develop rabbit antisera and/or rat and mouse monoclonal antibodies to the separate peptides, ii.) analyze by peptide mapping, in SDS gels or by HPLC, the 20kd and 70kd peptides, and iii.) scale up production of the alpha-LT to allow partial amino acid sequencing of the 70kd peptide in collaboration with Biogen. These studies will further characterize the human LT system at the peptide, mRNA, and genomic levels, and will generate elegant probes to assess the expression of LT gene(s) in activated T- and NK-effector cell populations.